Detection method and apparatus for detecting microbial growth

ABSTRACT

A system and method is provided for non-invasively measuring changes in a specimen suspected of containing one or more microbes, by monitoring changes in the dielectric constant of the specimen caused by metabolic processes of such microbes.

RELATED APPLICATION This application claims priority to U.S. ProvisionalPatent Application No. 60/639,366 filed Dec. 27, 2004, the disclosure ofwhich is hereby incorporated by reference. FIELD OF THE INVENTION

The present invention relates to a system and method to measure a changein a biological sample in a non-invasive manner to avoid contaminationof the sample.

BACKGROUND OF THE INVENTION

Currently, the presence of biologically active agents such as bacteriain a patient's body fluid, and especially in blood, is determined usingblood culture vials. A small quantity of blood is injected through anenclosing rubber septum into a sterile vial containing a culture mediumand the vial is then incubated and monitored for bacterial growth.

Common visual inspection of the culture vial then involves monitoringthe turbidity or observing eventual color changes of the liquidsuspension within the vial. Known instrument methods can also be used todetect changes in the carbon dioxide content of the culture bottles,which is a metabolic byproduct of the bacterial growth. Monitoring thecarbon dioxide content can be accomplished by methods well establishedin the art; however, most of these methods require invasive procedureswhich can result in the well-known problem of cross-contamination withinthe vial.

One solution to the above problems includes the use of a non-invasiveinfrared microorganism detection instrument in which special vialshaving infrared-transmitting windows are utilized. These vials, however,are relatively expensive. In yet another solution, glass vials aretransferred to an infrared spectrometer by an automated manipulator armand measured through the glass vial. The disadvantage of this system isthat, due to the high infrared absorption of glass, small changes in theglass wall thickness can generate large errors in the measured headspacegas absorption. These problems can be partly reduced by utilizinghigh-quality glass vials, but this measure results in relatively highvial cost.

Still other solutions have included the use of chemical sensors disposedinside the vial. These sensors respond to changes in the carbon dioxideconcentration in the liquid phase by changing their color or by changingtheir fluorescence intensity. These techniques are based on lightintensity measurements and require spectral filtering in the excitationand/or emission signals. However, in such solutions, errors can occur ifany of the light source, photodetector, filters, or sensor show agingeffects over time, which would vary the intensity response.

Accordingly, a need exists for a system and method to measure a changein a sample in a non-invasive manner to avoid contamination of thesample.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a system and method to measurethe changes in the dielectric constant of a biological sample in agrowth medium as microbes metabolize the essential components of themedium into metabolic byproducts.

In another embodiment, the invention provides a system and method forestablishing an electrodynamic field such that minute changes to abiological sample's dielectric constant through the destruction andformation of various organic and inorganic compounds will change acapacitance value. (As used herein, electrodynamic field indicates anelectric field set up such that physical or chemical changes inducemeasurable electrical changes, e.g., a varying electric field capable ofsensing a change in dielectric constant between capacitive electrodes).

In a further embodiment, the invention provides a system and method formeasuring a change in capacitance value and determining a correspondingchange value for a biological growth material.

According to an embodiment, a system and method is provided forgenerating an electrodynamic field on at least one and preferably aplurality of electrodes, positioned adjacent to a sample, and which havea suitable self-capacitance to virtual ground such that minutecapacitance changes within the sample can be measured. The electrodesare not required to be placed in direct contact with the sample, aschanges to the sample's dielectric constant through the destruction andformation of various organic and inorganic compounds will change thecapacitance of the electrodes to virtual ground, and provide ameasurement corresponding to the sample changes occurring.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages will be apparent uponconsideration of the following drawings and detailed description.Embodiments of the present invention are illustrated in the appendeddrawings, in which:

FIG. 1 is a schematic diagram of an embodiment of the invention;

FIGS. 2A and 2B illustrate a flexible circuit in accordance with anembodiment of the invention;

FIG. 3 is a non-invasive dielectric sensing apparatus in accordance withan embodiment of the invention;

FIG. 4 is a flowchart for a dielectric sensing method in accordance withan embodiment of the invention;

FIG. 5 is a graph illustrating an example of the capacitance changedetectable using a non-invasive dielectric sensing apparatus inaccordance with an embodiment of the present invention;

FIG. 6 illustrates another embodiment of the invention;

FIG. 7 illustrates a further embodiment of the present invention.

In the drawing figures, it will be understood that like numerals referto like elements.

DETAILED DESCRIPTION

One embodiment of the invention, an electrodynamic field is generatedusing at least one, and preferably a plurality of electrodes which arepositioned adjacent to a sample containing a biological sample that mayinclude microbes such as bacteria, typically in a growth media, theelectrodes having a suitable self-capacitance to virtual ground suchthat minute changes can be measured. The electrodes are not required tobe placed in direct contact with the sample, as changes to the sample'sdielectric constant through the destruction and formation of variousorganic and inorganic compounds will change the capacitance of theelectrodes to virtual ground. Typically, the entirety of the sample'sdielectric constant is measured, but it is possible to set up electrodesto monitor portions of the sample, e.g., a circumferential portion.While fluid samples in a growth media are typical, solid samples such asfood products or tissues are also contemplated.

In an embodiment of the invention shown schematically in FIG. 1, acircuit 100 comprising two conductive electrodes 102 and 104 is placedaround the circumference of an insulative growth vessel or bottle 106.In one aspect, the electrodes 102, 104 are configured in aninterdigitated format, as more clearly shown in FIG. 2A. Electrode 102can be configured as a sensor electrode, and electrode 104 can beconfigured as a ground reference electrode. The flexible circuit 100 istypically wrapped around the vessel or bottle 106, such that anelectrode surface area is in physical proximity to the liquid sample 108inside, where the dielectric constant of the sample 108 changes withmicroorganism growth and metabolite release.

The conductive sheets of both electrodes 102 and 104 are connected to adifferential alternating current signal generator 110 which produces asine wave to quantify circuit impedance. In the apparatus of FIG. 1, thesignal applied to one electrode is 180 degrees out of phase with thesignal applied to the other electrode. The frequency of the signal ischosen to provide a current from one electrode to the other throughtheir intercapacitance that can be easily quantified by standard methodsas known to those skilled in the art.

A resistor 112 is electrically coupled in series between the signalgenerator 110 and the sensor electrode 102, wherein the current variesthrough the resistor 112 at a rate which is inversely proportional tothe capacitance between electrodes 102 and 104. A test device 114, suchas a digital multimeter (DMM), can then be used to measure the voltagevalue across the resistor 112.

As noted above, the current varies through the resistor 112 inverselyproportional to the capacitance between electrodes 102 and 104. Thecapacitance between electrodes 102 and 104 can be illustrated by a modelcircuit 116, which illustrates the variables in free space capacitancebetween electrodes 102 Hand 104, between electrode 104 and ground, andbetween electrode 102 and ground. As known to those skilled in the art,capacitance varies between electrodes and other objects depending ontheir size, spacing and dielectric properties of the interveningmaterials (i.e., bottle 106 and the sample 108 inside).

FIG. 2A shows a configuration of the electrodes according to anembodiment of the invention. In FIG. 2A, the interdigitated electrodes102 and 104 are provided on or in a flexible circuit substrate 105, suchas mylar or kevlar film. In some embodiments, the electrodes 102 and 104are provided with contact tabs 101 and 103 to provide a means toelectrically couple each electrode with the signal generator 110. Anynumber of gaps or spacing can be provided between electrodes 102 and 104to establish a desired capacitance value. The flexible substrate 105 canthen be used to encircle the culture vial or bottle 106 as shown by FIG.2B. Once encircled, an electromagnetic field is established as apparentto one skilled in the art.

As the dielectric constant of the sample 108 inside the bottle 106changes, the current between the electrodes 102 and 104 changes in amanner that is inversely proportional to the dielectric constant changeof the sample bottle. This is a result of the change in capacitivereactance at the selected frequency. The dielectric constant changes forexample, when microorganisms within the sample 108 metabolize sugarsinto dissolved gases, enzymes, proteins, and waste products. Any numberof possible variables effecting the measurements can be evaluated, andthese are presented as examples only.

FIG. 3 shows an embodiment of a system where multiple bottles 106 can beinserted in an incubator assembly 302 (for convenience only a singlebottle 106 is shown). According to this embodiment, each bottle 108 isencircled by the flexible substrate and electrode of a flexible circuit100, e.g., of the type as described above. The encircled vials 106 canthen be fit within each incubator well 300 of the incubator assembly302. Within each well 300, electrical connectors can be provided (notshown) to receive the contact tabs 101 and 103 of each electrode 102 and104 disposed upon the flexible substrate of the flexible circuit 100.Once positioned, a method to measure changes in the sample, such as achange in the dielectric constant of a biological growth medium, can beimplemented as described herein.

An embodiment of the method of the invention is illustrated in the flowchart of FIG. 4. Specifically, once each bottle 106 is provided with aflexible circuit 100 and positioned within an incubator assembly 302, auser request can be made for detecting changes therein which can beachieved in the following steps 402 through 436. Upon a request todetermine a metabolite change in a bottle 106 at step 402, theembodiment of the present invention begins scanning the electrodynamicfield (e-field) sensor set (e.g., electrodes 102 and 104) at step 404.(In FIG. 4, “EF” refers to electric field reading and “En” refers toelectrode set number—one set per bottle). At this time, the system thenrecords the e-field sensor set data (EF₁) of a first bottle, and timestamps the measurement at step 406. Thereafter, at step 408 the systemmoves to the next e-field sensor set—En, records the e-field sensor setdata, and time stamps the measurement. The system continues to repeatsteps 406 and 408 until each set of e-field sensors has been measuredand recorded, and then returns to start when e-field sensors of all thebottles 106 have been measured.

Once a complete pass of all sensor sets has been completed in steps 406and 408, the system then stores the measurements in a record for thespecific sensor sets, respectively, at step 410. At step 412, the systemcan then evaluate sequential records for each sensor set. At step 416,the system can then detect changes in measured values, such as in thisexample, detecting if the current electric field reading shows anincrease of greater than 2% over each of the last three readings. Ifnot, the system evaluates the information at step 418 to determine ifthere is a decrease in the electric field reading of more than 10% overthe last five readings. If there is an increase at step 416, the systemthen determines if the last four electric field readings show a positivesecond derivative at step 420.

If the readings do not show a positive second derivative at step 420,then the system evaluates the information at step 422 to determine ifthe last four electric field readings show a negative second derivative.If there is a positive second derivative at step 420, the system thendetermines at step 424 if the increase in the current reading is greaterthan 90% of the value of the previous reading. If the increase of thecurrent reading is greater than 90% of the value of the previous readingat step 424, the system then determines that there is an indication ofpositive culture for the electrode at step 426. In other words, if thevalue of the current reading shows a value of signal strength at least90% or higher of the previous reading, a “positive” is declared. If thereading tested in step 424 is less than 90% of the previous reading, thesystem proceeds to step 428 to determine if the culture of the electrodebeing scanned has been tested for the minimum period as established bythe test protocol. If the test protocol period is satisfied, the systemdetermines an indication of negative culture at steep 432. If the testprotocol period has not been satisfied the system resumes scanning at402.

Returning to step 418, if there is a decrease in the electric fieldreading of more than 10% over the last five readings at step 418, thesystem proceeds to step 422. If not, the system evaluates theinformation at step 428 to determine if the culture of the electrode hasbeen tested for a minimum period as established by protocol. If theperiod is satisfactory, the system determines that there is anindication of negative culture for the electrode at step 432. In otherwords, if the value of the current reading shows no drop in signalstrength from the previous reading, a “negative” is declared. A lack ofdrop in the dielectric constant of the culture bottle's liquid contentscan indicate no metabolic process change has occurred. If the period isnot satisfactory, the system returns to step 412.

Returning to step 422, if the last four electric field readings show anegative second derivative at step 422, the system proceeds to step 426.A sudden precipitous drop in the dielectric constant of the culturebottle's liquid contents can indicate a metabolic process change by anymicroorganism present. For instance, the conversion from glucosemetabolism to catabolism. If not, the system then proceeds to step 428.Upon completion, of all sensor scanning for the entire length of eachculture's protocol, the system stops scanning the e-field sensors atstep 434, and enters a standby mode at step 436.

Variations in the above process are of course possible, depending on,for example, the parameters of the particular system or the particularsample type.

EXAMPLE

A BD BACTEC(tm) Plus Aerobic/F culture bottle was obtained. A single,flexible electrode of the type shown in FIG. 2A was assembled.Tin-coated copper adhesive tape was pre-cut to form the interdigitatedfingers and placed on a 0.005 inch thick Mylar substrate. Electricalconnections to the two electrodes were formed by two tabs extending fromthe fingers, and were connected to an electrical circuit board. Theelectrode was placed onto the bottle, to circumferentially surround it.

The electrode was attached to a Motorola 33794 Electric Field ImagingDevice evaluation module, which is a device designed for sensing objectswithin its self-generating electric field. Data was logged by a personalcomputer running a LabView “virtual instrument” application specificallydesigned for the 33794 device. The program periodically sent a digitalsignal to the 33794 via a serial data connection, and the integratedcircuit of the 33794 would measure the amount of field in the electrode,and send it to the computer for storage. The plot of FIG. 5 is arepresentation of that data. FIG. 5 shows the change in capacitance inthe bottle based on E. coli growth, having a vertical axis indicatingmeasured capacitance in microfarads, and a horizontal axis indicatingtime in seconds. At point 502, an inoculation of 0.1 ml of E. coli wasmade into the bottle. At point 504, approximately 26 minutes later, arise is shown in the capacitance measured by the electrodes. Thesaturation ends approximately 55 seconds later at point 506, marked by adecrease in the measured capacitance to a value at point 508. Thecapacitance returns to an equilibrium point 510 approximately 4 hourslater. These minute capacitance changes within the sample are measuredvia the electrodes and provide a measurement corresponding to the samplechanges occurring.

In addition to the bottles shown above, other containers can be usedaccording to embodiments of the invention. In the embodiment of FIG. 6,plates 605 and 607 include opposing pairs of conductive electrodes 602and 604, wherein each pair of electrodes are placed above and belowmicrotiter wells 606. Electrode 602 can be configured as a sensorelectrode, and electrode 604 can be configured as a ground referenceelectrode, substantially as described above in FIG. 1. The plates 605and 607 are positioned such that an electrode surface area is inphysical proximity to the microtiter wells 606, wherein the dielectricconstant of the microtiter wells 606 change with microorganism growthand metabolite release of samples located in the wells. The remainingfunctions and implementation of the electrodes 602 and 604, aresubstantially the same as those described above.

An embodiment of the invention useful with viscous or solid samples,such as food or tissue, is shown in FIG. 7. According to thisembodiment, the container consists of disposable compression plates 626to surround a sample 628. Electrode plates 622 and 624 can be providedas shown, having tabs 623 and 625, respectively. Conductive electrodeplates 622 and 624 are placed above and below sample compression plates626. Electrode 622 can be configured as a sensor electrode, andelectrode 624 can be configured as a ground reference electrode in aconfiguration such as shown in FIG. 1. The electrodes and plates arepositioned such that an electrode surface area is close enough to thesample 628 to allow measurement of the dielectric constant of the sample628 as it changes with microorganism growth and metabolite release. Theremaining functions and implementation of the embodiment of FIG. 7, aresubstantially the same as those described above.

Embodiments of the present invention can solve problems associated withvarying media fill density, varying vial geometry, container materialtransparency, mixture of solid, liquid, and semisolid components in thecontainer contents, and differences in the container material, whetherplastic or glass. The design of the interrogating electrodes can befurther used to determine the sensitivity and selectivity of the sensorto different components of the system.

The use of an electrodynamic field substantially avoids measurementproblems associated with variations in bottle sources, media types,resin fills, and artifact effects (foam, magnetic stirring element,settling, etc.). For example, effects such as foaming, presence orabsence of stirring element, blood settling, and presence/absence ofblood in the culture vessel generally do not affect measurements basedon changes in the electrodynamic field. Similarly, the container,vessel, vial or bottle bottom does not need to be perfectly flat orindented as shown in FIG. 1.

Embodiments of the present invention can be applied to a variety ofbiological sensing applications, such as infectious disease/antibioticsusceptibility testing, or the identification and antimicrobialsensitivity testing of particular microbiological isolates. Anotherapplication is the detection of contaminating organisms on food productsand pharmaceuticals.

Embodiments of the present invention can further be applied to thesensing of inorganic or organic chemical reactions that result in theformation of compounds that have a measurably different dielectricconstant than the original reactants. Variable initial permittivity thatis experienced in different media types due to their varying chemicalcomposition generally requires foreknowledge of the medium type to applythe correct interpretive algorithm.

Also, embodiments of the invention can be used to detect volume levelsin a container, as well as degree of agitation. For example, the fillvolume can be estimated to within 1 ml for a 60 ml container (<2%error). Thus, uses beyond growth detection are possible.

The invention is believed to be superior to other methods which detectthe growth of microbes because of a substantially increased sensitivity.For example, it has been shown that recovery of growth followingmicrobial stress can be detected within one hour for E. coli and S.epidermitis with the present invention.

In addition to the reduction in time to detect growth, it is believedthe invention is capable of detecting much lower concentrations oforganisms than current methods. An additional advantage provided by theembodiments of the present invention is that the measurements can betaken from outside the bottle electrically, and hence the container neednot be optically transparent.

Although only certain exemplary embodiments of the present inventionhave been described in detail above, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of the present invention. Accordingly, all such modificationsare intended to be included within the scope of this invention asdefined in the appended claims and equivalents thereof.

1. A system comprising: a container comprising a volume for holding aspecimen; and a circuit comprising: at least one electrode located incontact with or in proximity to the container; a signal generatorcapable of directing current through the at least one electrode suchthat an electrodynamic field forms over at least a portion of thevolume; and a test device capable of measuring a value of theelectrodynamic field.
 2. The system of claim 1, wherein the container iscylindrical or comprises a cylindrical portion and wherein the at leastone electrode is disposed circumferentially around the container.
 3. Thesystem of claim 1, wherein the system comprises two electrodes, the twoelectrodes being substantially planar and disposed on opposite sides ofthe container.
 4. The system of claim 2, wherein the at least oneelectrode comprises two interdigitated conductive elements, and whereina first of the two interdigitated conductive elements is a sensorelectrode and a second of the two interdigitated conductive elements isa ground reference electrode.
 5. The system of claim 1, wherein the atleast one electrode comprises two conductive elements, and wherein afirst of the two conductive elements is a sensor electrode and a secondof the two interdigitated conductive elements is a ground referenceelectrode.
 6. The system of claim 5, wherein the signal generator isconnected with the sensor electrode and the ground reference electrode,wherein the signal generator produces a sine wave, and wherein thesignal applied to the sensor electrode is 180° out of phase with thesignal applied to the ground reference electrode.
 7. The system of claim1, wherein the at least one electrode is disposed on an insulative,flexible substrate.
 8. The system of claim 1, further comprising aplurality of the containers and an incubator assembly comprising one ormore incubator wells for receiving the containers and at least a portionof the at least two electrodes, wherein the incubator wells compriseelectrical connections for the at least one electrode.
 9. The system ofclaim 1, wherein the container is a blood culture bottle comprising agrowth media.
 10. The system of claim 1, wherein the container comprisesthe specimen, and wherein the specimen is a biological specimensuspected of containing one or more microbes.
 11. The system of claim10, wherein the system is capable of monitoring changes in the specimencaused by metabolic processes of the one or more microbes.
 12. Thesystem of claim 11, wherein the changes in the electrodynamic field arecapacitance changes indicative of changes in the dielectric constant ofthe specimen.
 13. A process for detecting changes in a specimen,comprising the steps of: providing a container comprising a specimensuspected of containing one or more microbes; providing at least oneelectrode in contact with or in proximity to the container; generatingan electrodynamic field, using the at least one electrode, over at leasta portion of the specimen; and detecting changes in the electrodynamicfield, wherein the changes are capacitance changes indicative of changesin the dielectric constant of the specimen.
 14. The process of claim 13,wherein the changes are caused by metabolic processes of the one or moremicrobes.
 15. The process of claim 13, wherein the container is a bloodculture bottle comprising a growth media and a blood sample.
 16. Theprocess of claim 13, wherein the step of detecting changes comprises:measuring a value of the electrodynamic field and recording the time andthe value; repeating the measuring and recording step one or more times;and evaluating the records.
 17. The process of claim 16, furthercomprising the steps of providing a plurality of the containers andperforming the measuring, recording and repeating steps for each of thecontainers.
 18. The process of claim 16, wherein the step of evaluatingthe records comprises applying a set of queries to the records todetermine if the specimen is positive or negative for microbe growth.19. The process of claim 13, wherein the at least one electrodecomprises at least two electrodes.